Journal: Nucleic Acids Research

Article Title: Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

doi: 10.1093/nar/gkw792

Figure Lengend Snippet: nvGW182 represses tethered mRNA via W-motifs. ( A ) Sequence of the nvGW182 CED (nvCED) with tryptophans in presumptive W-motifs shown in bold. ( B ) Mutations of W residues (W→A) in W-motifs alleviate repression by the nvCED. Plasmids encoding either wt NHA-nvCED or its indicated mutants were co-transfected into HEK293 cells, together with RL-5BoxB and FL. As negative control, untethered HA-nvCED was used. Mutants 1W1A through 11W11A contain up to eleven mutated W residues (for details, see Materials and Methods). Schematic representations of nvCED constructs are drawn below the corresponding bars. Asterisks indicate presumptive W-motifs; W residues are shown in black and W→A mutations in red. Data were analyzed and presented as in Figure (means ±SD, n = 4–10). Expression of NHA-fusion proteins was estimated by Western blotting. ( C ) W-motifs additively contribute to repression by tethered nvCED. Experiment was performed as in (B), except that nvCED with four N-terminal W-motifs mutated were used (n = 4). ( D ) W-motifs contribute to repression by the full-length nvGW182. Experiment was performed as in (B), except that full-length nvGW182 was used instead of nvCED domain (n = 4–12).

Article Snippet: Human /Nematostella GW182 chimeric constructs were generated by PCR-based fusion of shRNA-resistant TNRC6A N-terminal region (aa 1–1169, Q8NDV7-2) with nvCED (aa 1159–1698), wt or 11W11A and cloning the resulting hybrid coding sequences into pCiNeo-NHA (deposited to Addgene 82430, 82431) and pEBG vector (deposited to Addgene 82432, 82433).

Techniques: Sequencing, Transfection, Negative Control, Construct, Expressing, Western Blot

Journal: Nucleic Acids Research

Article Title: Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

doi: 10.1093/nar/gkw792

Figure Lengend Snippet: nvGW182 recruits subunits of the CCR4-NOT complex via its W-motifs. ( A ) Quantitative mass spectrometric analysis of proteins associated with nvGW182. The GST-nvGW182 full-length constructs, the nvCED domain only, as wild type and 11W11A mutant variant, were expressed in HEK293 cells and affinity purified using GST. GST alone was used as a negative control. On the right, relevant interactors for each variant are listed along with their corresponding enrichment factor (the log2 ratio of label-free quantification values (LFQ intensity) over corresponding LFQs detected in pull-down of GST tag alone). On the left, volcano plot showing proteins with differential binding to nvCED and nvCED11W11A variant. The logarithmic ratios of protein intensities were plotted against negative logarithmic P -values of a two samples t-test (two sided). A hyperbolic curve separates significantly enriched proteins from common binders. Selected interactors recruited via W-motifs of nvCED are labeled in red (FDR ≤ 0.05, n = 2). ( B ) Validation of CCR4-NOT-nvCED interactions identified in (A). GST-fusions of indicated proteins were expressed in HEK293 cells, and inputs (6%) and GST-pulldowns were analyzed by Western blotting using the indicated antibodies. Human TNRC6CΔCED was used as positive control for AGO2 binding. TNRC6C CED and TNRC6C 7W7A CED were used as positive and negative controls for CNOT7 and CNOT9 binding. Asterisks (*) indicate unspecific bands. ( C ) W-motifs function additively to recruit the CCR4-NOT complex. The experiment was performed as in (B), except that nvCED with four N-terminal (N-4W4A) or C-terminal (C-4W4A) W-motifs mutated were used for pull-downs. nvCED and nvCED 11W11A were used as positive and negative controls for CNOT7 binding, respectively. Schematic representations of nvCED domains are shown below the corresponding lane. Asterisks indicate W-motifs; W residues are shown in black and W→A mutations in red.

Article Snippet: Human /Nematostella GW182 chimeric constructs were generated by PCR-based fusion of shRNA-resistant TNRC6A N-terminal region (aa 1–1169, Q8NDV7-2) with nvCED (aa 1159–1698), wt or 11W11A and cloning the resulting hybrid coding sequences into pCiNeo-NHA (deposited to Addgene 82430, 82431) and pEBG vector (deposited to Addgene 82432, 82433).

Techniques: Construct, Mutagenesis, Variant Assay, Affinity Purification, Negative Control, Quantitative Proteomics, Binding Assay, Labeling, Biomarker Discovery, Western Blot, Positive Control

Journal: Nucleic Acids Research

Article Title: Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

doi: 10.1093/nar/gkw792

Figure Lengend Snippet: nvGW182 represses translation and mediates mRNA degradation via CNOT6/7 deadenylases. ( A ) The deadenylation catalytic activity of the CCR4-NOT complex contributes to nvGW182-mediated silencing. HEK293 cells were co-transfected with RLuc-boxB, FLuc, tethered NHA-nvGW182 or NHA-nvCED, and increasing amounts of CNOT6 and CNOT7 catalytic mutants (CNOT cat ). Untethered HA-fusions were used as negative controls (means ±SD, n = 3-6). ( B ) Interfering with the CCR4-NOT deadenylation activity leads to stabilization of nvGW182-bound mRNAs. RLuc and FLuc mRNA from nvGW182-tethering experiments shown in (A) were estimated by qRT-PCR. Filled bars: +CNOT cat ; open bars: -CNOT cat . RLuc values were normalized to FLuc mRNA. Untethered HA-fusions were used as negative controls (means ±SD, n = 2). ( C ) nvGW182 silences tethered mRNA via translational repression and stimulation of mRNA decay. RLuc and FLuc mRNA and protein levels from nvGW182-tethering experiments were estimated by qRT-PCR (RNA, open bars) and luciferase assays (protein, filled bars). Data were normalized and presented as in (A and B).

Article Snippet: Human /Nematostella GW182 chimeric constructs were generated by PCR-based fusion of shRNA-resistant TNRC6A N-terminal region (aa 1–1169, Q8NDV7-2) with nvCED (aa 1159–1698), wt or 11W11A and cloning the resulting hybrid coding sequences into pCiNeo-NHA (deposited to Addgene 82430, 82431) and pEBG vector (deposited to Addgene 82432, 82433).

Techniques: Activity Assay, Transfection, Quantitative RT-PCR, Luciferase

Journal: Nucleic Acids Research

Article Title: Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution

doi: 10.1093/nar/gkw792

Figure Lengend Snippet: Human/ Nematostella GW182 chimera partially rescues miRNA silencing in human HeLa cells. ( A ) Schematic representation of human /Nematostella GW182 protein chimeras (h/nvGW182) used in (B) and (C). AGO-binding N-terminal region of human TNRC6A was fused to nvCED, either wild type or 11W11A mutant. ( B ) h/nvGW182 chimera binds endogenous human AGO2. GST-fusions of the indicated proteins and their domains were expressed in HEK293 cells, and inputs (6%) and GST-pulldowns were analyzed by Western blotting using the indicated antibodies. ( C ) W-motifs of h/nvGW182 chimera are required to rescue depletion of endogenous TNRC6 proteins. HeLa cell line carrying stably integrated inducible shRNAs construct against endogenous TNRC6A and B was used for TNRC6 knockdown experiments (open bars); HeLa cells not expressing TNRC6-directed shRNAs was used as a control (filled bars). Expression of shRNAs was induced for 2 days prior to transfection of miRNA reporters and rescue constructs. Cells were transfected with RLuc-hmga2 reporter containing let-7 sites or its mutant version (RLuc-hmga2 mut), and increasing amounts of plasmids expressing h/nvGW182 chimera or its 11W11A mutant. TNRC6A was used as a positive control, TNRC6A 8W8A was used as a negative control. Values represent percentages of RLuc activity produced by hmga2-mut reporter without TNRC6 depletion (means ±SD, n = 3). ( D ) Speculative model illustrating the mechanisms of miRNA-mediated silencing in bilaterian animals (recruitment of the CCR4-NOT complex via W-motifs of GW182), land plants (slicing by AGO and translational repression via an unknown mechanism that depends on AGO), and cnidarians (both slicing and GW182-dependent mechanism). The close-up shows the graphic representation of the W-motif that recruits the deadenylation complexes and is conserved in human, Drosophila and Nematostella . The consensus was derived with the MEME suite , see also Supplementary Figure S1.

Article Snippet: Human /Nematostella GW182 chimeric constructs were generated by PCR-based fusion of shRNA-resistant TNRC6A N-terminal region (aa 1–1169, Q8NDV7-2) with nvCED (aa 1159–1698), wt or 11W11A and cloning the resulting hybrid coding sequences into pCiNeo-NHA (deposited to Addgene 82430, 82431) and pEBG vector (deposited to Addgene 82432, 82433).

Techniques: Binding Assay, Mutagenesis, Western Blot, Stable Transfection, Construct, Knockdown, Expressing, Control, Transfection, Positive Control, Negative Control, Activity Assay, Produced, Derivative Assay